# Origin of replication discovery for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products

## TL;DR

Researchers found new replication origins in bacteria, enabling the production of foreign proteins and compounds in a non-model bacterium.

## Contribution

Identification of three new origins of replication functional in gram-negative bacteria, including one validated in Pantoea sp. MT58.

## Key findings

- A barcoded plasmid library identified three new origins replicating in gram-negative bacteria.
- Origin 6911 replicated with a copy number of 9 in Pantoea sp. MT58.
- Plasmids with origin 6911 produced GFP, indigoidine, and isoprenol in Pantoea sp. MT58.

## Abstract

Leveraging predicted origin sequences from a previously characterized groundwater plasmidome, we constructed a barcoded plasmid library to screen for previously unknown origins. Testing this library against a panel of representative bacterial strains led to the identification of 3 previously unknown origins that replicate in gram-negative bacteria not previously associated with these origin sequences. Experimental validation confirmed that a plasmid bearing origin 6911 as the sole origin could replicate with a copy number of 9 (±2) in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium. Plasmids based on this new origin were used to express the reporter protein GFP, and non-native metabolite pathways for the natural product indigoidine and the terpenoid compound isoprenol. Functional previously unknown origins of replication in such non-model organisms can expand the toolkit for genetic manipulations of both model and less-studied bacteria.

•Plasmid library constructed with known and predicted bacterial origins of replication•Discovery of 3 previously unknown origins that replicate in gram-negative bacteria•Origin validation in Pantoea sp. MT58 through copy number determination•Bioproduction of GFP, indigoidine, and isoprenol in Pantoea sp. MT58

Plasmid library constructed with known and predicted bacterial origins of replication

Discovery of 3 previously unknown origins that replicate in gram-negative bacteria

Origin validation in Pantoea sp. MT58 through copy number determination

Bioproduction of GFP, indigoidine, and isoprenol in Pantoea sp. MT58

Molecular biology; Molecular genetics; Molecular mechanism of gene regulation; Molecular microbiology